Radio-wave responsive doped nanoparticles for image-guided therapeutics

ABSTRACT

The invention discloses nanoparticles comprising compounds of calcium with anions such as phosphate, pyrophosphate, sulphate, silicate, carbonate, molybdate, or phosphosilicate that are doped with various ions. The nanoparticles are configured to produce heat (hyperthermia) under radio-wave (1 KHz-1000 GHz) exposure together with magnetism suitable for contrast imaging in MRI, X-ray absorption for computed tomography, near-infrared optical fluorescence for optical imaging, and/or radio-isotope emission for nuclear imaging or therapy. The nanoparticles can also be incorporated into micro-beads or other 3 dimensional scaffolds for image-guided (MRI, CT, NIR, nuclear) tissue regeneration, immunotherapy, vascular or tumor embolization, and/or chemo/radio-embolization.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2016/064890, filed on 5 Dec. 2016, which claims priority to Indianpatent application No. 6495/CHE/2015, filed on 3 Dec. 2015, the fulldisclosure of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to image guided therapeutics and inparticular to nanoparticle compositions that produce heat orhyperthermia on exposure to alternating radiofrequency waves, withsimultaneous visibility under multiple imaging mechanisms.

DESCRIPTION OF THE RELATED ART

Radiofrequency ablation (RFA) is a treatment technique used in theclinics for treating tumors, cardiac problems, pain and varicose veins.It is based on the principle of hyperthermia for the destruction ofdiseased tissues. In clinics, RFA is administered by placing one or moreelectrodes in the diseased tissue and the application of alternatingcurrent or field in the radiofrequency range. Heating takes place in thetissue near to the emitting electrodes. Hyperthermia therapy refers toapplication of temperatures in the range of 40-50° C. while applicationof temperatures above 50° C. is referred to as thermo ablation. Themajor limitation of current RFA is that the size of the lesion that canbe treated is limited to 4 cm or less. In clinics, this problem isaddressed by repeated repositioning of RF electrodes in order to coverthe entire disease area. Recent developments such as expandable andinternally cooled electrodes have helped to increase the area ofablation to a small extent. In addition, normal saline infusion has beenshown to be effective in enlarging the area of necrosis duringradiofrequency ablation, by improving the conductivity 3-5 times greaterthan that of blood and 12-15 times greater than that of soft tissues.Currently RFA is done under ultrasound guidance that provides poorcontrast for the diseased tissue. RF probe can be applied moreaccurately if the contrast of the diseased tissue is enhanced by MRI, CTor PET/SPECT-CT.

Calcium phosphosilicate nanoparticles have been developed for nearinfrared imaging and drug delivery applications (WO2011057216 A1).Calcium phosphate nanoparticles have also been disclosed forencapsulating photosensitizers for PDT (EP 2 198 885 B1) and fordelivery of therapeutics to tumor cells and lymphatics for treatment ofcancer and prevention of metastasis (US 2011/0318422 A1). Theapplication of iron oxide incorporated calcium phosphate nanoparticleshas been disclosed for applications such as magnetic hyperthermia,imaging, drug delivery (US 2014/0044643). A few other references discussthe development of doped apatite and calcium phosphate nanoparticles fordifferent combinations of optical, MRI, X-ray, nuclear and/or ultrasoundimaging (U.S. Pat. No. 5,342,609, U.S. Pat. No. 5,690,908, WO 2011151631A1, US 2010/0092364 A1).

The present invention is directed at nanoparticle formulations that aidin locating lesions through visualization by various methods incombination or independently, while simultaneously providing localizedablation of disease cells under radiofrequency wave irradiation, withattendant advantages as set forth herein.

SUMMARY OF THE INVENTION

Radio-wave responsive particle formulations that are simultaneouslyuseful for multiple modes of imaging and therapy are disclosed. Theformulations comprise an anion-cation complex represented as D-CX,wherein C is the calcium cation, X is an anion selected from phosphate,pyrophosphate, sulphate, silicate, phosphosilicate, molybdate andcarbonate. D is a dopant selected from one or more of Mo, Bi, Ba, Sr,Se, Ta, Cd, W, I, Zr, Ta, Hf, Au, Ag, Cu, Zn, Si, Fe, Mn, Al, Pt, Ce,Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Pm, Sm, Sc, Tb, Tm, Yb or Y. Theparticles are micro- or nano-particles in the size range of 1-1000 nm.The complex is configured to generate heat under exposure toradiofrequency (RF) waves.

The particle formulation is further configured to provide simultaneousT1 and T2 contrast under magnetic resonance imaging (MRI), provide X rayabsorption for CT imaging, provide near infrared (NIR) fluorescence foroptical imaging, or emit radiation for nuclear imaging.

In some embodiments of the particle formulation the complex isbeta-tricalcium phosphate (Ca₃(PO₄)₂), calcium dihydrogen phosphate(Ca(H₂PO₄)₂), calcium hydrogen phosphate (CaHPO₄), monocalcium phosphatemonohydrate (Ca(H₂PO₄).H₂O), dicalcium phosphate dihydrate(CaHPO₄.2H₂O), tetracalcium phosphate (Ca₄(PO₄)₂O), octacalciumphosphate (Ca₈H₂(PO₄).5H₂O), fluorapatite (Ca₅(PO₄)₃F), chlorapatite(Ca₅(PO₄)₃Cl), or a combination thereof. In some embodiments the complexis calcium sulphate (CaSO₄), calcium hydrogen sulphate (Ca(HSO₄)₂),calcium sulphate dihydrate (CaSO₄·2H₂O), calcium sulphate hemihydrate(CaSO₄·5H₂O), or a combination thereof. In some embodiments the complexis calcium carbonate (CaCO₃), calcium bicarbonate (Ca(HCO₃)₂), hydratedcalcium carbonate (CaCO₃·nH₂O, or a combination thereof. In oneembodiment the complex is calcium molybdate (CaMoO₄). In someembodiments the complex is a calcium silicate comprising 3CaO·iO₂,2CaO·SiO₂, 3CaO·2SiO₂, CaO·SiO₂, 3CaO·2SiO₂·4H₂O, CaO·Al₂O₃SiO₂,Ca₃SiO₅, Ca₃Si₂O₇ or a combination thereof. In some embodiments thecomplex is a calcium phosphosilicate comprising 35-65 wt % SiO₂, 1-50 wt% Na₂O, 10-90 wt % CaO, and 1-50 wt % P₂O₅.

In some embodiments of the formulation, the heat generated is up to 100°C. on exposure to a radiofrequency field of frequency ranging from 1Hz-100 GHz and power in the range 1-1000 W for a time period rangingfrom 0.1 seconds to 1 hour.

In various embodiments, the complex is configured to providesimultaneous T1 and T2 contrast in magnetic resonance imaging (MRI) dueto doping with D at a level varying from 0.0001 to 50 atomic % of thecalcium (Ca²⁺). The dopant D in these embodiments comprises ions of Fe,Mn, Eu, Tb, Se, Er, Dy, Ho, Tm, Al, Mo, Ag, Au, Cu, Zn, Si, orcombinations thereof.

In some aspects, the formulations are further configured to providenear-infrared fluorescence emission at the 650-1000 nm spectral region,by doping (D) with an organic molecule selected from indocyanine greenor fluorescene, at levels from 0.0001 to 50 weight % of the complex.

In some aspects the formulations are further configured to providenuclear contrast for one or more of single photon emission computedtomography, positron emission tomography (SPECT/PET) or radionuclidemediated therapy by surface labelling with a radionuclide selected from¹⁵³Sm, ^(99m)Tc, ¹²³I, ¹⁸F, ¹³¹I, ¹¹¹In, ¹⁸⁸Re, ¹⁶⁶Ho, ⁹⁰Y, ⁸²Rb, ²²⁵Ac,²¹¹At, ²¹²Bi, ²¹³Bi, ²²³Ra, ²¹²Pb, ²²⁷Th, or ¹⁴⁹Tb.

In other aspects, the formulations are further configured to provide CTcontrast by doping with at least one impurity selected from molybdenum,bismuth, barium, strontium, tantalum, cadmium, tungsten, iodine,zirconium, tantalum, hafnium, lanthanum, gold, iron, aluminium, platinumor combinations thereof.

In various aspects, the formulation is selected from one of(Ca,xFe,yMo)₃ (PO₄)₂, (Ca,xFe,yMo)₁₀(PO₄)₆(OH)₂), (Ca, xFe,yMo)₂SiO₄, or(Ca,xFe,yMo)NaO₆PSiO₄ where x varies from 0-50 wt %, y varies from 0-30wt % and wherein the formulation is configured to provide T1-T2 MRcontrast together with X-ray CT contrast.

In some aspects the particles have spherical or non-spherical shape withsize ranging from 1 nm to 2000 nm. In some aspects the particles arefurther co-loaded with one or more therapeutic agents forradio-wave-triggered controlled drug release. In some embodiments theformulation is a radio-wave responsive, MR, CT, nuclear and/or NIRimageable micro-bead formulation ranging in size from 1 μm to 1 mm forvascular embolization or tissue implantation. In some embodiments theformulation is a radio-wave responsive, MR, CT, nuclear and/or NIRimageable micro-bead formulation labelled with radioisotopes forradio-embolization therapy. In some embodiments the formulation is aradio-wave responsive, MR, CT, nuclear and/or NIR imageable formulationfor culturing, proliferating, differentiating, activating, orreprogramming biological cells for therapeutics.

A method of image guided radiofrequency treatment of cancer using aparticle formulation in a subject with cancer is disclosed. Theparticles are micro- or nano-particles in the size range of 1-1000 nm.The method comprises administrating to the subject, particles comprisingone of beta-tricalcium phosphate (Ca₃(PO₄)₂), calcium dihydrogenphosphate (Ca(H₂PO₄)₂), calcium hydrogen phosphate (CaHPO₄), monocalciumphosphate monohydrate (Ca(H₂PO₄)·H₂O), dicalcium phosphate dihydrate(CaHPO₄·2H₂O), tetracalcium phosphate (Ca₄(PO₄)₂O) or octacalciumphosphate (Ca₈H₂(PO₄)·5H₂O), fluorapatite (Ca₅(PO₄)₃F), chlorapatite(Ca₅(PO₄)₃Cl), calcium sulphate (CaSO₄), calcium sulphate dihydrate(CaSO₄·2H₂O) and calcium sulphate hemihydrate (CaSO₄·5H₂O), calciumcarbonate (CaCO₃), calcium molybdate (CaMoO₄), 3CaO·SiO₂, 2CaO·SiO₂,3CaO·2SiO₂, CaO·SiO₂, 3CaO·2SiO₂·4H₂O, CaO·Al₂O₃·2SiO₂, Ca₃SiO₅,Ca₃Si₂O₇, a calcium phosphosilicate with compositions ranging from 35-65wt % SiO₂, 1-50 wt % Na₂O, 10-90 wt % CaO, 1-50 wt % P₂O₅, Ca,xFe,yMo)₃(PO₄)₂, (Ca,xFe,yMo)₁₀(PO₄)₆(OH)₂), (Ca, xFe,yMo)₂SiO₄,(Ca,xFe,yMo)NaO₆PSiO₄ or combinations thereof. The nanoparticles may bedoped with a dopant selected from a chloride, bromide, iodide, fluoride,nitrate, sulphate, carbonate or oxide salt of a metal selected from oneor more of Mo, Bi, Ba, Sr, Ta, Se, Cd, W, I, Zr, Ta, Hf, La, Au, Fe, Al,Pt, Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Pm, Sm, Sc, Tb, Tm, Yb, orY, or a dye comprising indocyanine green or fluorescene, and conjugatedwith an agent comprising one or more of a radiolabel, a bisphosphonatedrug, a capping agent or a targeting ligand. The method then involvescontacting the particles with the cancer cells and then imaging theparticles-containing cancer cells by one or more of magnetic resonance,nuclear, near infrared, or computed tomography to detect the location ofthe cancer cells. A therapeutic dose of radiofrequency waves is thenapplied to the cancer cells to ablate the cells and RF-induced deliveryof the conjugated agent from the particles to the cancer cells mayadditionally be effected.

The method of treatment in some aspects may further comprisesimultaneous or separate dual mode (T1-T2) MR, nuclear imaging-guidedcell culture and tissue regeneration. In some aspects the method mayfurther comprise nuclear imaging-guided radio-chemoembolization andradiation therapy. In further aspects the method may comprise imageguided modulation, activation, suppression, re-programming, editing ofcells and immunotherapy for treating a disease condition. In someaspects the method may comprise cancer detection and therapy. In someaspects the method may comprise stimulating, treating, carrying anddelivering, one or more of stem cells, induced pluripotent stem cells,differentiated cells, immune cells, bacteria, or viruses.

The method of treatment in some embodiments may further comprise imageguided delivery of a therapeutic agent comprising a chemodrug, siRNA,DNA, RNA, a peptide, a protein, or a gene. In various embodiments thetreatment may comprise administrating the particles subcutaneously,orally, intravenously or intraperitoneally, or implanting as gels, beadsor scaffolds. In various embodiments the particles activate immuneresponse against the cancer cells in the subject.

This and further aspects are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention has other advantages and features which will be morereadily apparent from the following detailed description of theinvention and the appended claims, when taken in conjunction with theaccompanying drawings, in which:

FIG. 1 illustrates in one embodiment a system of radio-wave responsiveparticles for tumour ablation.

FIG. 2 is a schematic of a method of preparing radio-wave responsiveparticles for tumour ablation.

FIG. 3 illustrates exemplary methods of treatment using RF-responsiveparticles for tumour ablation.

FIG. 4A schematic of Fe-nCX (X=phosphate) where a few Ca²⁺ atoms arereplaced by doped Fe³⁺.

FIG. 4B TEM image showing particles of size ˜10 nm.

FIG. 5 shows Zeta potential data peaked at ˜15 mV for Fe³⁺ doped calciumphosphate nanoparticles.

FIG. 6A shows VSM data and FIG. 6B shows magnetic susceptibility data ofdifferent batches of Fe-nCX (X=phosphate) doped with varyingconcentration of Fe³⁺.

FIG. 7A shows T2 and T1 weighted MR contrast of 1.25 mg/mL of Fe-nCX(X=phosphate) doped with varying concentration of Fe³⁺, dispersed inagar phantom.

FIG. 7B and FIG. 7C show r2 and (FIG. 7C) r1 relaxivity plot of 4.1 at %Fe³⁺ doped Fe-nCX respectively.

FIG. 8A and FIG. 8B show in vivo (FIG. 8A) T1 weighted and (FIG. 8B) T2weighted MRI of Wistar rat before and after Fe-nCX (X=phosphate)injection.

FIG. 8C and FIG. 8D show enhancement of both T1 and T2 contrast aftersample injection (FIG. 8C) axial liver section before sample injection,and (FIG. 8D) axial liver section 30 minutes after sample injection.FIG. 8E shows T2 mapping data averaging 3 ROI selected in the axialsections before and 1 hr after injection

FIG. 9A T2 weighted MRI of wistar rat over a period of 96 hours afterintravenous injection of Fe-nCX (X=phosphate), FIG. 9B showscorresponding axial liver sections over a period of 96 hrs.

FIG. 9C illustrates variation in T2 time of liver over a period of 96hours after Fe-nCX injection and FIG. 9D shows Fe content in differentorgans estimated by ICP analysis.

FIG. 10A shows T2 weighted MR image (coronal section) of subcutaneoustumour before sample injection, while FIG. 10B shows T2 weighted andFIG. 10C shows T1 weighted MR image of subcutaneous tumour afterintratumoral Fe-nCX (X=phosphate) injection.

FIG. 11 shows impedance spectroscopy results illustrating radio waveresponsiveness of doped nanoparticles.

FIG. 12 depicts radiofrequency assisted heating of nanoparticles Fe-nCX(X=phosphate) at concentrations varying from 10-500 μg/ml at 100 W RFpower for 1 minute.

FIG. 13A shows RF response of C6 glioma cells treated with varyingconcentrations of Fe-nCX (X=phosphate) for 4 hours and FIG. 13B showsradio-wave responsive nanoparticles injected into rat liver tumor modelwith enhanced hyperthermal ablation (81.3° C.) compared to untreatedanimal (51.3° C.).

FIG. 14A is a photograph of undoped CP beads and FIG. 14B is SEM imageof doped CP bead showing size ˜1 μm (D) T1 and T2 weighted MRI of theGd-nCX (X=phosphate) beads.

FIG. 15A is a photograph of phantom bone with small defect filled withGd-nCX (X=phosphate), FIG. 15B is T1 weighted MRI of the phantom bonedefect filled with Gd-nCX beads, sagittal section, and FIG. 15C is axialT1 weighted MRI with bright T1 weighted contrast visible from Gd:nCXbeads.

FIG. 16A shows coronal, sagittal and transaxial images using CTcontrast, FIG. 16B shows nuclear contrast of the Mo-nCX (X=phosphate)microbeads in a cotton phantom and FIG. 16C shows fused CT-nuclearimages confirming the findings.

FIG. 16D shows drug release data at different RF power applied ofdoxorubicin incorporated into Mo-nCX beads.

FIG. 17A shows proliferation assay of rat mesenchymal stem cells treatedwith Fe-nCX (X=phosphate), FIG. 17B illustrates rMSC treated with 100μg/mL of Fe-nCX for 6 hours, FIG. 17C shows MRI of cell pellet ofunlabelled rMSC and Fe-nCX labelled rMSC, FIG. 17D illustrates MRI ofrat brain injected with unlabelled cells and FIG. 17E is MRI of ratbrain injected with labelled cells, with injected regions marked.

FIG. 18 is MRI of coronal section of orthotopic rat liver tumor modelbefore sample injection (left) and after Fe-nCX (X=phosphate) injection(right), showing tumor margins more clearly demarcated after injection.

FIG. 19A is MRI of cells growing in Fe-nCX (X=phosphate) scaffold seenas white spots growing through the Fe-nCX incorporated scaffold, FIG.19B is SEM image of Fe-nCX incorporated scaffold and FIG. 19C is SEMimage of cells growing on Fe-nCX scaffold.

FIG. 20A and FIG. 20B are SEM images of nanoparticle uptake bymacrophages, representing immune cells.

FIG. 20C shows FACS analysis of cytokine induction of macrophage afterPBS treatment and FIG. 20D is FACS analysis showing increased cytokineinduction by nanoparticle treated macrophages.

DETAILED DESCRIPTION OF THE EMBODIMENTS

While the invention has been disclosed with reference to certainembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the scope of the invention. In addition, manymodifications may be made to adapt to a particular situation or materialto the teachings of the invention without departing from its scope.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein unless the context clearlydictates otherwise. The meaning of “a”, “an”, and “the” include pluralreferences. The meaning of “in” includes “in” and “on.” Referring to thedrawings, like numbers indicate like parts throughout the views.Additionally, a reference to the singular includes a reference to theplural unless otherwise stated or inconsistent with the disclosureherein.

The invention in its various embodiments discloses nanoparticlescomprising doped phosphate, sulphate, phosphosilicate, or bioactiveglass compounds of calcium, termed as ‘D-nCX’, where D represents dopantions, n indicates nanometer size, C is calcium, X is anions such asphosphate, pyrophosphates, sulphate, silicate, carbonate, molybdate, orphosphosilicate. The nanoparticles produce heat (hyperthermia) underradio-wave (1 KHz-1000 GHz) exposure together with providing magnetismsuitable for contrast imaging in MRI, X-ray absorption for computedtomography, near-infrared optical fluorescence for optical imaging,and/or radio-isotope emission for nuclear imaging or therapy. Thenanoparticles can also be incorporated into micro-beads or other 3dimensional scaffolds for image guided (MRI, CT, NIR, nuclear) tissueregeneration, vascular or tumor embolization, and/orchemo/radio-embolization. The nanoparticles or associated systems canalso be used for image guided drug delivery, gene delivery, siRNAdelivery, stem cell labeling, activation, suppression or re-programmingof immune cells.

The invention in one embodiment proposes a system 100 of calcium-basedcompounds D-CX that can be formed into nanoparticle formulations asdisclosed in FIG. 1. System 100 comprises the complex D-CX calcium (alsodenoted as C) as the cation 101 reacted with an anionic group 102,denoted as X in the description. The system comprises dopant 103,intended to provide various types of functionality to the formulations(also denoted as D). In various embodiments, the anionic group 102 isselected from phosphate, pyrophosphate, sulphate, silicate,phosphosilicate, molybdate and carbonate. Dopant 103 is a metal selectedfrom one or more of Mo, Bi, Ba, Sr, Ta, Cd, W, I, Zr, Ta, Hf, La, Au,Fe, Al, Pt, Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Pm, Sm, Sc, Tb, Tm,Yb or Y. The particulate system 100 is configured to generate heat underradiofrequency wave exposure.

In various embodiments the system 100 is also configured to provideother functionalities for imaging such as simultaneous T1 and T2contrast under magnetic resonance imaging (MRI), X ray absorption for CTimaging, near infrared (NIR) fluorescence for optical imaging or emitradiation for nuclear imaging. The functionalities are provided byincorporating a suitable combination of dopants 103 in the formulation.

In various embodiments the complex of system 100 is a phosphate ofcalcium such as beta-tricalcium phosphate (Ca₃(PO₄)₂), calciumdihydrogen phosphate (Ca(H₂PO₄)₂), calcium hydrogen phosphate (CaHPO₄),monocalcium phosphate monohydrate (Ca(H₂PO₄)·H₂O), dicalcium phosphatedihydrate (CaHPO₄·2H₂O), tetracalciumphosphate (Ca₄(PO₄)₂O),octacalciumphosphate (Ca₈H₂(PO₄)·5H₂O), fluorapatite (Ca₅(PO₄)₃F),chlorapatite (Ca₅(PO₄)₃Cl), or combinations thereof. In some embodimentsthe complex 100 is a sulphate of calcium such as calcium sulphate(CaSO₄), calcium hydrogen sulphate (Ca(HSO₄)₂), calcium sulphatedihydrate (CaSO₄·2H₂O), calcium sulphate hemihydrate (CaSO₄·5H₂O), orcombinations thereof. In various embodiments the complex 100 is acarbonate of calcium such as calcium carbonate (CaCO₃), calciumbicarbonate (Ca(HCO₃)₂), hydrated calcium carbonate (CaCO₃·nH₂O, or acombination thereof. In one embodiment the complex 100 is calciummolybdate (CaMoO₄). In some embodiments the complex 100 is a calciumsilicate such as 3CaO·SiO₂, 2CaO·SiO₂, 3CaO·2SiO₂, CaO·SiO₂,3CaO·2SiO₂-4H₂O, CaO·Al₂O₃·2SiO₂, Ca₃SiO₅, Ca₃Si₂O₇, or a combinationthereof. In some embodiments the complex 100 is a calciumphosphosilicate or bioglass comprising 35-65 wt % SiO₂, 1-50 wt % Na₂O,10-90 wt % CaO, and 1-50 wt %/P₂O₅.

In various embodiments the complex 100 is configured to respond to RFwave exposure by heating. In some embodiments the heat generated is upto 100° C. on exposure to a radiofrequency field of frequency rangingfrom 1 Hz-100 GHz. The power applied may be in the range 1-1000 W for atime period ranging from 0.1 seconds to 1 hour.

In various embodiments where the particles are intended for ablatingcancer tissue, the particles incorporating the complex 100 areconfigured to be nanoparticles having spherical or non-spherical shapewith size ranging from 1 nm to 2000 nm. In some embodiments where theparticles are intended to be implanted into a human or animal body fortissue regeneration, the particles could be microparticles with sizeranging from a few microns to a few mm.

In some embodiments the complex 100 is configured to providesimultaneous T1 and T2 contrast in magnetic resonance imaging (MRI). Inembodiments in which simultaneous T1 and T2 contrast is provided, theparticles carry dopant D at a level varying from 0.0001 to 50 atomic %of the calcium (Ca²⁺) ion content. Dopant D comprises ions of Fe, Mn,Eu, Tb, Er, Dy, Ho, Tm, Al, Mo, Ag, Au, Cu, Zn, Si, or combinationsthereof.

In some embodiments the complex 100 is configured to providenear-infrared (NIR) fluorescence emission in the 650-1000 nm spectralregion. The NIR emission is provided by doping (D) with an organic dyemolecule. The organic dye could be any dye that provides fluorescence inthe relevant spectral range. The dopant could be selected fromindocyanine green or fluorescene at levels from 0.0001 to 50 weight % ofthe complex.

In some embodiments the complex 100 is further configured to provide CTcontrast by doping with suitable species. The CT contrast is provided bydoping with an impurity ion such as molybdenum, bismuth, barium,strontium, tantalum, cadmium, tungsten, iodine, zirconium, tantalum,hafnium, lanthanum, gold, iron, aluminium, platinum or combinationsthereof.

In various embodiments the complex 100 is further configured to providenuclear contrast by emission of radiation. The emission is provided toenable detection of the complex 100 using techniques such as singlephoton emission computed tomography or SPECT, positron emissiontomography or PET, or radionuclide mediated therapy. In variousembodiments the nanoparticles or microparticles of the invention aretagged for nuclear contrast by surface labelling with a radionuclideselected from ¹⁵³Sm, ^(99m)Tc, ¹²³I, ¹⁸F, ¹³¹I, ¹¹¹In, ¹⁸⁸Re, ¹⁶⁶Ho,⁹⁰Y, ⁸²Rb, ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ²²³Ra, ²¹²Pb, ²²⁷Th, or ¹⁴⁹Tb.

In some embodiments, the complex 100 is a radio-wave responsive, MR, CT,nuclear and/or NIR imageable micro-bead formulation ranging in size from1 μm to 1 mm for vascular embolization or tissue implantation. Inalternate embodiments the complex 100 may be further functionalized forradio-embolization therapy. In some embodiments the complex 100 may besurface-conjugated with a chemodrug, siRNA, DNA, RNA, a peptide, aprotein, a gene or a gene fragment.

In various embodiments, the invention discloses in FIG. 2 methods 200for preparing the nanoparticle formulations 100 disclosed in FIG. 1. Themethod 200 involves in step 201, adding and mixing precursor solutionsPart A containing Ca ions, Part B containing anion species, Part Ccontaining hydroxyl ions (OH⁻, a surfactant solution in Part D and anaqueous solution containing a dopant and additionally a dye in Part E toa container and reacting them (step 202) to form the precipitateparticles. Further, in step 203, the precipitate particles are allowedto grow by aging. The reacting step 202 and the aging step 203 may becarried out for 1-24 hours at a temperature in the range 25-120° C. Thenext step 204 involves washing the precipitate particles withde-mineralized water to remove solutes and reaction products. Theparticles are thereafter calcined (step 205) at a temperature in therange 200-1500° C. to obtain dry powder. In a further step 206, the drypowder particles are reconstituted using water or phosphate buffersaline and surface-conjugated with radiolabels, bisphosphonate drugs,capping agents or targeting ligands to provide additionalfunctionalities depending on the application, to obtain the particleformulation.

In various embodiments the solution containing calcium ions in Part A isprepared from water soluble, miscible or dispersible salts of calciumhydroxide, chloride, bromide, iodide, fluoride, nitrate, sulphate,carbonate or oxide.

In various embodiments, the precursor compound in Part B containing ananion, is formed from a species selected from phosphate, pyrophosphate,sulphate, carbonate, molybdate, silicate, and phosphosilicate.

For forming calcium phosphate nanoparticles, the precursor solution forphosphate anions in Part B in various embodiments is formed from a watersoluble or miscible salt of a phosphate. The phosphate ion source couldbe a phosphate of sodium—Na₃PO₄, Na₂HPO₄, NaH₂PO₄, potassium—K₃PO₄,K₂HPO₄, KH₂PO₄, lithium—Li₃PO₄, Li₂HPO₄, LiH₂PO₄, ammonium—(NH₄)₃PO₄,(NH₄)₂HPO₄, NH₄H₂PO₄ or alternatively, phosphoric acid, or a combinationthereof.

For forming calcium sulphate nanoparticles, the precursor solution forsulphate anions in Part B in various embodiments is prepared usingsulphuric acid or sodium sulphate salt or a combination thereof.

To prepare calcium carbonate nanoparticles, the precursor solutioncontaining carbonate ions in Part-B in various embodiments isconstituted of a soluble carbonate salt or carbonic acid. The carbonatesalt could be sodium carbonate, potassium carbonate or ammoniumcarbonate, or combinations thereof.

To prepare calcium molybdate nanoparticles, the precursor solutioncontaining molybdate ions in Part B in various embodiments could beconstituted from a soluble salt of molybdenum. The salt could be sodiummolybdate, potassium molybdate, ammonium molybdate, or combinationsthereof.

To prepare calcium silicate particles, the anion source of Part B invarious embodiments could be either silica or a soluble silicate salt.The soluble silicate salt could be sodium silicate, potassium silicate,calcium silicate, or combinations thereof.

In various embodiments, the precursor compound containing hydroxylanions in Part C is formed by a hydroxide salt of either sodium,potassium, lithium, ammonium or calcium.

To prepare calcium phosphosilicate or bioglass particles, Part-B invarious embodiments includes silica, tetraethyl orthosilicate (TEOS) ora soluble silicate salt, while the solution in Part C includes aphosphate source. The soluble silicate salt could be sodium silicate,potassium silicate, or combinations thereof. The phosphate source inPart-C could be triethyl phosphate, diammonium hydrogen phosphate,sodium monophosphate, sodium diphosphate, sodium polyphosphate,phosphorus pentoxide, or combinations thereof.

In some embodiments the surfactant solution in Part D, is formed by anaqueous solution of monosodium, disodium or trisodium citrate or citricacid. In some embodiments the solution may additionally include apolymer such as polyethylene glycol, poly-L lactic acid, polyethyleneimine, or poly(lactic-co-glycolic acid).

In various embodiments, the dopant ions in Part E are configured toprovide various functionalities such as T1 and T2 magnetic contrast,X-ray absorption for CT imaging, or near-infrared fluorescence foroptical imaging, at a level varying from 0.0001 to 50 atomic % of thecalcium (Ca²).

In some embodiments the T1 and T2 magnetic contrast is provided byincluding in Part E a solution containing ions such as manganese (II),iron(II), iron (III), gadolinium (III) or other lanthanides such as Dy,Er, Eu, Ho, La, Lu, Nd, Pr, Pm, Sm, Sc, Tb, Tm, Yb or Y, or Al, Mo, Ag,Au, Cu, Zn, or Si, or combinations thereof. In further embodiments, theCT contrast is provided by adding in Part E a solution containing ionsof molybdenum, bismuth, barium, strontium, tantalum, cadmium, tungsten,iodine, zirconium, tantalum, hafnium, lanthanum, gold, iron, aluminium,or platinum or combinations thereof. In another embodiment, Part E couldfurther comprise a solution containing dye molecules that givesnear-infrared fluorescence to the nanoparticle. The dye molecules couldbe NIR emitting dyes such as indocyanine green (ICG) or fluorescene.

In some embodiments the doped nanoparticle is surface conjugated duringthe reconstitution step 206 of the method to provide therapeutic effectsor radiolabelling or other therapeutic property. In some embodimentssurface labelling with a radionuclide selected from ¹⁵³Sm, ^(99m)Tc,¹²³I, ¹⁸F, ¹³¹I, ¹¹¹n, ¹⁸⁸Re, ¹⁶⁶Ho, ⁹⁰Y, ⁸²Rb, ²²⁵Ac, ²¹¹At, ²¹²Bi,²¹³Bi, ²²³Ra, ²¹²Pb, ²²⁷Th, or ¹⁴⁹Tb is done through ligands such asbisphosphonates. The radionuclide labelling is intended to providenuclear contrast for a technique such as single photon emission computedtomography or SPECT, positron emission tomography (PET) or radionuclidemediated therapy. In some embodiments the nanoparticles may be co-loadedwith therapeutic agents for radio-wave-triggered controlled drugrelease.

The invention further comprises methods of treatment using the system100 disclosed with reference to FIG. 1 and method of preparation withreference to FIG. 2. In some embodiments, methods of treatment 300 asdisclosed in FIG. 3 comprise the step 301 of administering to a patientwith cancer a particle formulation 100 containing a calcium compounddoped with a metal ion, a targeting ligand and a dye, functionalized asillustrated in previous embodiments. In various embodiments the step 301may comprise administrating the particles subcutaneously, orally,intravenously or intraperitoneally. In some embodiments in step 301 theymay also be implanted as gels, beads or scaffolds. In the next step 302,the particles are contacted with the cancer cells. The target area isthen visualized (step 303) by imaging the particles-containing cancercells by a method such as magnetic resonance, nuclear, near infrared, orcomputed tomography to detect the location of the cancer cells. Thevisualization in step 303 may be simultaneous or separate dual mode(T1-T2) MR, nuclear imaging or optical imaging using the functionalityprovided by system 100. In the next step 304, RF ablation is provided ina therapeutic dose to effect ablation to the contacted cancer cells.Concurrently or subsequently, in step 305, the RF wave exposure may alsotrigger release of one or more conjugated agents, markers or otherspecies to the cancer cells.

Currently, in clinics, separate contrast agents are being used for T1and T2 weighted imaging. Gadolinium complexes are the most widely usedT1 contrast agents whereas super-paramagnetic iron oxide nanoparticles(SPIONS) are used as T2 contrast agents. Usually T1 weighted imaging iscarried out for anatomical imaging purposes whereas T2 weighted imagingis carried out for obtaining pathological or functional details. Thetreatment modalities provided by the system 100 according to embodimentsof the method of treatment 300 are further illustrated. In oneembodiment of the method 300, the unique advantage of the inventiveparticles of system 100 is that using this single system therapists canimage a diseased condition utilizing both T1 and T2 (bright and dark) MRcontrast and also enhance the RF response of the diseased tissue. In analternate embodiment of the method 300, the therapists can also switchover to X ray contrast using the same system because the samenanoparticles can give both contrast simultaneously or separately withMRI, as the particles 100 may be co-doped with X ray absorbing ions. Ineffect, embodiments of the method 300 disclosed herein provide formulti-modal image guided RF hyperthermia therapy.

In some embodiments of the method 300, in addition to MRI or CTcontrast, the particles can also be used to provide near-infraredcontrast for optical imaging. Near infrared contrast is achieved byco-doping organic dyes such as indocyanine within the particles 100together with magnetic or CT contrast dopants to provide additionalproperty of near-infrared fluorescence emission.

In other aspects of the method 300, bisphosphonate conjugated nuclearlabels, as illustrated in earlier embodiments, for example99Technitium-MDP, are efficiently tagged on the calcium atoms of D-nCXnanoparticles through calcium-phosphonate linkage, to provide nuclearcontrast. Combinatorial imaging using MRI-CT together with nuclearmethods like SPECT-PET is then done to provide simultaneous highdefinition anatomical, physiological and functional information aboutthe diseases like cancer and image guided application of multipleradio-wave mediated processes. The radio-wave mediated processes couldbe one or more of hyperthermia, drug-delivery, gene delivery, or othertherapeutics. The system 100 and the method 300 in the variousembodiments not only provide contrast imaging but also provideradio-wave responsiveness that is useful for multitude of applicationssuch as hyperthermia, RF triggered therapeutic release, cell activation,tissue regeneration etc.

In further aspects of the method 300, the nanoparticles are used to formmicro-beads of size varying from 1-1000 microns for image guided (dualmode MRI, CT, NIR and/or nuclear) embolization, chemo-embolization orradio-embolization of tumor or vasculature. For example, the method 300may comprise embolization of the feeding artery to a tumor. Theprocedure of embolization is done by injecting micro-beads through atrans-arterial catheterization under the guidance of X-ray CT or MRI.The excellent X-ray and MR contrast of the disclosed system 100 provideunique advantages over existing agents and can be loaded withinpolymeric micro-beads for use in embolization therapy. The samemicrobeads can also be surface conjugated or labeled with therapeuticradio-nucleotides for image guided nuclear medicine or embolization.

In yet other aspects of the method 300, the micro-beads formed from D-CXnanoparticles are co-loaded with chemodrugs, siRNA, therapeuticpeptides, proteins, small molecules, genes, etc. to conduct RF-triggereddrug release. Due to the radio-wave hyperthermia response of theembedded nanoparticles, the microbeads or scaffolds are configured toexpand and contract with respect to the application of RF wave andthermal-energy leading to the triggered drug release. In alternativeembodiments, these nanoparticles can be co-loaded into drug loadedthermo-responsive polymeric nanoparticles such that with exposure to RFwave, the increase in temperature triggers the drug release.

In various embodiments of the method 300, these nanoparticles of thesystem 100, owing to their MR, X-ray, NIR contrast, are used for imageguided tissue regeneration. In some embodiments, these nanoparticles areloaded into porous micro-beads or 3D polymeric scaffolds where stemcells, or differentiated organ cells or tissues are regenerated and thesame are monitored in vivo using different imaging modalities such asMRI or CT. The RF responsivity of nanoparticles can be used to stimulatethe cells for better proliferation and differentiation into varioustissues and phenotypes.

In various aspects of the method 300, the RF responsive nanoparticles ofsystem 100 are conjugated with immunotherapeutic molecules such ascancer antigens, peptides or small molecules and used as image guidedimmunotherapeutic adjuvant or immune cell stimulating/suppressingagents. Under RF exposure, these nanoparticles may stimulate thecontrolled or triggered delivery of immunologic agents together withproducing heat energy at local tissue regions that may attract immunecells to the site of heating and hence externally controllable immuneresponse may be obtained for various therapeutic scenarios such ascancer or autoimmune disease.

In other embodiments of method 300, the system 100 is labelled with stemcells or other types of cells for their image guided delivery andactivation in vivo. For example, human mesenchymal stem cells can belabelled with MR-CT imageable D-CX nanoparticles and injected into thedisease site or intravenously such that the kinetics of the cells can bemapped using MRI or CT. Additionally, owing to the RF response, theinjected cells can be activated using external RF trigger. For example,an antigen or peptide released from the radio-responsive NPs under RFexposure may trigger immune cell activation of differentiation of stemcells.

While the invention has been disclosed with reference to certainembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted withoutdeparting from the scope of the invention. In addition, manymodifications may be made to adapt to a particular situation or materialthe teachings of the invention without departing from its scope asfurther explained in the following examples, which however, are not tobe construed to limit the scope of the invention as delineated by theclaims

EXAMPLES Example—1 Preparation of Iron Doped Calcium PhosphateNanoparticles (nCP: Fe) for Dual T1-T2 Magnetic Contrast GuidedRadiofrequency Ablation of Tumor—Synthesis and Characterization

20 mL of 0.5 M calcium chloride (CaCl₂, Sigma, USA) was mixed with 20 mLof 0.2 M trisodium citrate (Na₃C₆H₅O₇, Fisher Scientific, India) and 0.1M FeCl₃ (Sigma, USA). Volume of 0.1 M FeCl₃ added was varied as per therequired percentage of doping. 5 mL of 0.3 M diammonium hydrogenphosphate ((NH₄)₂HPO₄, S.D Fine Chemicals, India) mixed with 0.2 mL of 3N ammonium hydroxide (NH₄OH, Fisher Scientific, India) was added dropwise to the above mixture of CaCl₂, Na₃C₆H₅O₇ and FeCl₃ under constantstirring to obtain Fe doped calcium phosphate nanoparticles, Fe-nCX(X=phosphate). The precipitate was washed 4 times in hot distilled waterby centrifugation at 8500 rpm for 15 minutes and redispersed in PBS.FIG. 4A shows the schematic of Fe-nCX, wherein Fe³⁺ replaces Ca²⁺ in thecalcium phosphate crystal lattice. Successful doping of Fe³⁺ within nCPis clearly indicated by the yellow color of Fe-nCX solution. TEM showsuniform size distribution of ˜10 nm (FIG. 4B). The efficiency of Fe³⁺doping, estimated by ICP analysis, showed that on an average ˜40% of theadded ions were doped within nCP (FIG. 5A). In order to investigate ifdoped Fe³⁺ was leaching out of Fe-nCX, the final supernatant obtainedafter fourth wash was analyzed. Fe³⁺ level in supernatant was below thelevel of detection by ICP that indicated Fe³⁺ is efficiently dopedwithin nCP matrix. The zeta potential of Fe-nCX was estimated to be −15mV (FIG. 5B) which is attributed to presence of citrate ions on thesurface of Fe-nCX. Magnetic property of Fe-nCX analyzed by VSM, showedlinear increase in magnetization with external field indicatingparamagnetic behavior (FIG. 6A) compared to diamagnetic property ofundoped nCP. The paramagnetic nature of Fe-nCX is conferred by Fe³⁺ dueto the presence of a lone unpaired electron in its 3d orbital. Asexpected, increase in Fe³⁺ doping concentration from 0.5 to 6 wt %resulted in enhancement of the paramagnetic behavior (FIG. 6A) andmagnetic susceptibility (FIG. 6B).

Increase in Fe³⁺ dopant concentration from 1.5 to 6 wt % resulted inenhancement of T2 contrast intensity (FIG. 7A) whereas T1 contrastincreased up to a concentration of 4.1 wt % after which it reduced. Thuswe identified that at an optimum doping concentration of 4.1 wt %,Fe-nCX provided an efficient dual mode T1/T2 contrast in MRI. 4.1 wt %Fe doped nCP will be hereafter mentioned as Fe-nCX. Mapping studies werecarried out to estimate the T1 and T2 relaxivity values. FIG. 7B, 7Cshow variation in T2 and T1 relaxation time with increase innanoparticle concentration of Fe-nCX. T1 relaxivity value (r1) of Fe-nCXwas estimated as 0.75 mM⁻¹s⁻¹ and T2 relaxivity value (r2) as 29.6mM⁻¹s⁻¹. Although the obtained r1 and r2 values are lower than that ofclinically available contrast agents (r1 value Gd-DOTA33 at 7 T is 2.8mM⁻¹s⁻¹, r2 value of FeO at 1.5 T is 213 mM⁻¹s⁻¹), considering theintratumoral injection of Fe-nCX, the obtained contrast will besufficient for clinical applications.

Preparation of Chitin Nanogels (CN):

Chitin solution was prepared by adding chitin to saturated CaCl₂solution in methanol and dissolving by vigorous stirring using overheadstirrer for nearly 48 h at room temperature. 0.05% chitin solution wasprepared by using this method and they were further used for thepreparing nCN by employing controlled regeneration chemistry and wetmilling methods. The preparation and characterization of this controlnCN were done using usual methods

Example 2—Demonstration of In Vivo Dual Mode T1 and T2 Contrast

1 hour after Fe-nCX(X=phosphate) injection, an enhancement of both T1and T2 contrast was observed especially in the liver and heart region(shown in white dotted box: FIG. 8A, 8B). Axial T2 weighted images ofliver sections before (FIG. 8C) and after (FIG. 8D) Fe-nCX injectionclearly showed enhancement in T2 contrast intensity after sampleinjection that was also reflected in T2 mapping data (FIG. 8E) obtainedfrom selected ROI (white circles in FIG. 8B, 8C). Reduction in T2contrast in liver was associated with reduction of T2 relaxation timefrom 40 ms to 25 ms. To evaluate the biodistribution of Fe-nCX, MRI ofsample injected animal was carried out over a period of 96 hours. T2weighted whole body coronal (FIG. 9A) and axial (FIG. 9B) MRI showed anincrease in T2 contrast in liver (shown in white dotted box) within thefirst one hour which gradually reduced to initial contrast by 96 hours.This variation was also reflected in the T2 relaxation values thatdrastically reduced in the first hour after injection beyond which itgradually increased to initial value in ˜96 hours (FIG. 9C). This wasdue to the initial RES mediated accumulation of Fe-nCX within liver thatgradually got metabolized or cleared over a period of 96 hours. This wasconfirmed by ICP analysis which showed that 1 hour after the sampleinjection, there was an increase in Fe3+ content in all organs as wellas plasma with significantly higher concentration in the heart, plasmaand liver (FIG. 9D). These results suggest that the nanoparticlescirculated throughout the body for ˜1-2 hours during which it slowlyaccumulated in the liver before being cleared or metabolized throughhepatobiliary route.

To test the ability of Fe-nCX to provide dual T1-T2 contrast onintra-tumoral injection, 10 mg/kg of sample was injected to subcutaneoustumor (C6 glioma) in Wistar rat. Excellent T2 and T1 contrast wasobserved from the sample injected regions (shown in white box: FIG.10A-10C)

Example 3: In Vitro Radiofrequency Response of Fe-nCX (X=Phosphate)

RF response of the nanoparticles was measured in a custom madenon-invasive 13.5 MHz RF instrument. Different concentrations of Fe-nCXvarying from 10-500 μg/mL were taken in a small glass petri dish and 100W RF power was applied for 1 minute. The temperature of the solution wasmeasured before and after RF irradiation. In the frequency range appliedfor RF ablation (350-550 kHz), there was an increase in dielectric lossfactor, tan delta value, from 2.4 for undoped nCP to 3.54 for Fe-nCX(FIG. 11), that indicates lossy character of doped sample under RFexposure. We compared the RF mediated heating of nCP and Fe-nCX atvarying concentration at 100 W RF power for 1 minute exposure in anon-invasive RF machine. In the concentration range of 50-500 μg/mL,there was significant increase in temperature of up to 22° C. for Fe-nCXsamples compared to only <10° C. for undoped nCP (FIG. 12). We presumedthat this rise in temperature observed for Fe-nCX samples would besufficient to enhance efficiency of RFA under in vivo conditions.

RF response of nanoparticle treated N1-S1 hepatoma cells was also testedusing the same non-invasive RF instrument. N1-S1 cells were seeded in 24well plates at a seeding density of 2.5×10⁴ cells/well. Differentconcentrations of Fe-nCX (50-500 μg/mL) were added to the wells. Afterincubation with the nanoparticles for 4 hours, the cells are irradiatedwith 100 W RF power for 5 minutes. 4 hours after RF treatment, media waschanged. After 48 hours, cell viability analysis was carried out usingAlamar blue assay. There was a significant reduction in viability forcells treated with Fe-nCX compared to undoped nCP (FIG. 13A). Inuntreated control 40% of the cells were live after RF treatment. 300μg/mL of undoped nCP reduced viability to ˜31% whereas the sameconcentration of Fe-nCX reduced cell viability to 5%. This results werealso repeated in animal models where we could see an increasedtemperature of 81 degree Celsius in D-nCP treated animal liver tumor(FIG. 13 B, top panel) compared to 51° C. in untreated animal (FIG. 13B, lower panel).

Example 4: Method of Using D-nCX (X=Phosphate) Nanoparticles for DualMode MR or Dual Mode MR-CT Contrast Based Evaluation of Bone TissueRegeneration—Preparation of Doped Calcium Phosphate-Alginate Beads withMR Contrast

Iron Doped nCP Synthesis (Fe-nCX—X is Phosphate):

20 mL of 0.5 M calcium chloride (CaCl₂, Sigma, USA) was mixed with 20 mLof 0.2 M trisodium citrate (Na₃C₆H₅O₇, Fisher Scientific, India) and 0.1M FeCl₃ (Sigma, USA). Volume of 0.1 M FeCl₃ added was varied as per therequired percentage of doping. 5 mL of 0.3 M diammonium hydrogenphosphate ((NH₄)₂HPO₄, S.D Fine Chemicals, India) mixed with 0.2 mL of 3N ammonium hydroxide (NH₄OH, Fisher Scientific, India) was added dropwise to the above mixture of CaCl₂, Na₃C₆H₅O₇ and FeCl₃ under constantstirring to obtain Fe-nCX. The precipitate was washed 4 times in hotdistilled water by centrifugation at 8500 rpm for 15 minutes andredispersed in PBS.

Preparation of Fe-nCX-Alginate Composite Beads:

10 mL of 3 wt % alginate solution was prepared and kept for stirring for20 minutes until the alginate is completely solubilized. 60 wt % ofFe-nCX was added to the alginate solution and blended using mortar andpestle or a blender (IKA, US). The blended alginate-Fe-nCX was addeddrop wise to 1 wt % calcium chloride solution to form Fe-nCX-alginatebeads. The beads were strained, washed thrice with distilled water anddried in hot air oven at 60° C. overnight.

Characterization of Beads:

SEM image showed smooth surface morphology with size ˜1 μm (FIG. 14A) T1weighted and T2 weighted MRI of the beads showed dual contrastproperties (FIG. 14B). The capability of Fe-nCX-alginate beads toprovide contrast when placed in a bone defect was tested in a phantombone sample (FIG. 15A). Defect of size ˜1 cm was made in pig bone andfilled with beads as shown in FIG. 15A. MRI showed bright T1 contrastfrom the beads (FIG. 15B, 15C).

Example 5: Doped Calcium Phosphate Nanoparticles for Image GuidedImmunotherapy—Synthesis of Bisphosphonate (Zoledronic Acid) Loaded DopedCalcium Phosphate Nanoparticles for Activation of T Cells

20 mL of 0.5 M calcium chloride (CaCl₂, Sigma, USA) was mixed with 20 mLof 0.2 M trisodium citrate (Na₃C₆H₅O₇, Fisher Scientific, India) and 0.1M FeCl (Sigma, USA). Volume of 0.1 M FeCl₃ added was varied as per therequired percentage of doping. 5 mL of 0.3 M diammonium hydrogenphosphate ((NH₄)₂HPO₄, S.D Fine Chemicals, India) mixed with 0.2 mL of 3N ammonium hydroxide (NH₄OH, Fisher Scientific, India) was added dropwise to the above mixture of CaCl₂, Na₃C₆H₅O₇ and FeCl₃ under constantstirring to obtain Fe-nCX (X=phosphate). The precipitate was washed 4times in hot distilled water by centrifugation at 8500 rpm for 15minutes and redispersed in PBS. Zoledronic acid (1 mg/mL) was added toFe-nCX solution and incubated at room temperature (22-37° C.) for 30minutes. The zoledronic acid loaded Fe-nCX was then washed twice withdistilled water to obtain the final product.

Example 6: CT and Nuclear Contrast of Radio-Sensitive Nanoparticles

Synthesis of Molybdenum Doped Calcium Phosphate Nanoparticles (Mo-nCX, Xis Phosphate) and Demonstration of CT Contrast Property:

15 ml of 0.5M calcium chloride (CaCl₂, Sigma, USA) was mixed with 0.1Mammonium molybdate ((NH₄)₆Mo₇O₂₄.4H₂O, Nice Chemicals, India). Volume ofammonium molybdate was varied according to the required percentage ofdoping. 5 ml of 0.3M diammonium hydrogen phosphate ((NH₄)₂HPO₄, S.D FineChemicals, India) was mixed with 3N ammonium hydroxide (NH₄OH, FisherScientific, India) and added drop-wise to the reaction mixture, underconstant stirring. Precipitate washed 5 times with distilled water andcentrifugation at 7000 rpm for 10 minutes.

The CT contrast of the nanoparticles was assessed using the GE HawkeyeSPECT-CT system (GE Healthcare, USA). The contrast/attenuation providedby the nanoparticles was quantified with Hounsfield units (HU). Thehighest HU was obtained with 50% molybdenum doping.

Method of Combined CT and Nuclear Contrast Property of Microbeads Madeof Molybdenum Doped Calcium Phosphate Nanoparticles:

1% sodium alginate (Sigma, USA) solution was prepared and under constantstirring the molybdenum doped calcium phosphate nanoparticles (80% w/wof sodium alginate) was slowly added slowly and kept for stirring atroom temperature for 2 hours. Using a micro pipette, thisalginate-nanoparticle mixture was dropped into 2% (w/v) calcium chloride(Fisher Scientific, India) solution to produce the microbeads. The beadswere removed after 2 hours form the CaCl₂ solution, washed 5 times withdistilled water and dried for 24 hours in a hot-air oven (60° C.). 30mCi of 99m Technetium Methylene Diphosphonate (tracer) was added to 50mg of the microbeads and incubated at room temp for 2 hours. The excessuntagged tracer was washed using distilled water. FIG. 16A, depicts theCT contrast offered by the beads which were placed in a cotton phantom.FIG. 16B shows the corresponding nuclear contrast from the same beads.The same GE imaging system was used for the evaluation. The fused hybridimages (nuclear and CT) in FIG. 16C confirm the beads to simultaneouslyhave both CT and nuclear contrast. After application of RF of differentpower, the doxorubicin loaded microbeads shows controlled drug release.(FIG. 16D).

Example 7: Method of RF Triggered Drug Release from Microbeads PreparedUsing RF Responsive Calcium Phosphate Coloaded with Doxorubicin

1% sodium alginate (Sigma, USA) solution was prepared. Under constantstirring the Mo-nCX (X is phosphate) (80% w/w of sodium alginate) andDoxorubicin (Dox) (0.5% w/w of sodium alginate) were slowly added slowlyand kept for stirring at room temperature for 2 hours. Using a micropipette, this mixture was dropped into 2% (w/v) calcium chloride (Fisherscientific, India) solution to produce the microbeads. The beads wereremoved after 2 hours form the CaCl₂ solution, washed 5 times withdistilled water and dried for 24 hours in a hot-air oven (60° C.). Tostudy the drug release, multiple samples of 5 mg of the dried beadssuspended in 25 ml of phosphate buffered saline were prepared andmaintained at 37° C. Each of the samples was exposed to uniform RF(13.56 MHz) field of 5 W, 10 W and 15 W power for 1 minute, and the drugreleased into PBS immediately after exposure was measured using a UVspectrometer, at 488 nm (FIG. 16 D).

Example 8: Method of Labelling Stem Cells with Fe-nCX (X=Phosphate) andits MR Guided Tracking in Brain

Fe-nCX was used for labelling rat mesenchymal stem cells (rMSC) andtracking stem cell migration after injection to rat brain. rMSC wereisolated from rat femur bone marrow. The effect of nanoparticle taggingon the proliferation of rMSC was investigated for a period up to 7 days.It was observed that there was no change in proliferation of labelledcells compared to unlabelled rMSC (FIG. 17A). This confirmed that thenanoparticles do not affect the proliferative capability of the stemcells. 100 μg/mL of Fe-nCX was treated with these cells for a period of12 hours. FIG. 17B shows the Prussian blue stained nanoparticle withinrMSc after incubation for period of 12 hours. For MRI of cell pellet,100 μg/mL of Fe-nCX was treated with these cells for a period of 12hours. The untagged nanoparticles were washed with PBS, trypsinised andcentrifuged to form pellet. MRI of the labelled cell pellet showed T2based dark contrast compared to unlabelled cells (FIG. 17C). T2 value ofunlabelled cell pellet was 121 mS and that of labelled cells was 67 mS.The reduction in T2 value clearly shows the T2 shortening effect ofFe-nCX. 10⁵ Fe-nCX labelled rMSC were injected into the rat brain andtracked upto a period of 7 days. MRI of the rat brain clearly showed theinjected cells (FIG. 17E) compared to control brain injected withunlabelled cells (FIG. 17D).

Example 9: Method of Using the Fe-nCX (X=Phosphate) Nanoparticles forCancer Detection

Fe-nCX was used for the identification of small liver tumor inorthotopic rat liver tumor model. 10 mg/kg of the nanoparticles wasinjected to orthotopic rat liver tumor model and MRI was carried outafter 10 minutes. As seen in FIG. 18, the small liver tumor margin wasclearly demarcated after Fe-nCX injection (FIG. 15B). This is due to theaccumulation of the nanoparticles in the normal liver region (uptake bykupffer cells in healthy liver region) that enhances the relativecontrast of the tumor.

Example 10. Method of Using Fe-nCX (X=Phosphate) NanoparticlesContaining Scaffold for Tissue Regeneration

Fe-nCX scaffold provides an enhanced T2 contrast compared to undopedscaffold due to the T2 shortening by Fe³⁺ions. Cell ingrowth and tissueregeneration into the scaffold can be evaluated using MRI. As shown inFIG. 19A, the white spots seen in the Fe-nCX scaffold are cells growinginto the scaffold. The undoped scaffold shows a bright contrast and thecells cannot be spotted in the undoped scaffold. FIG. 19B shows SEMimage of the scaffold showing its macro porous nature facilitating cellgrowth and proliferation. FIG. 19C shows mesenchymal stem cell growingon the scaffold. The morphology of the attached cells showed that thescaffold is compatible to the mesenchymal stem cells.

Example 11: Delivery D-nCX to Immune Cells to Stimulate or CytokineRelease

In this example we showed that the said nanoparticles can be deliveredto immune cells like macrophages in large concentrations as shown inFIGS. 20A and 20B. The spherical particles are D-nCX taken up by themacrophages. This delivery caused release of pro-inflammatory cytokinesfrom the immune cells (macrophages) as shown in the flow cytometry data(FIG. 20 C (Before treatment) and FIG. 20 D (after treatment).

What is claimed is:
 1. A radio-wave responsive particle formulation,comprising: a doped anion-cation complex represented as D-C X, wherein:C is calcium cation (Ca²⁺); X is an anion selected from the groupconsisting of phosphate, pyrophosphate, and phosphosilicate; and D is adopant comprising 0.0001 to 50 atomic % of Mo and 1.5 to 6 atomic % ofFe, relative to Ca²⁺; wherein the complex is configured to: generateheat under exposure to radiofrequency (RF) waves; provide simultaneousT1 and T2 contrast under magnetic resonance imaging (MRI); and provide Xray contrast under CT imaging, wherein the particle formulationcomprises particles that are nano- or micro-particles having a size in arange of 1-2000 nm.
 2. The particle formulation of claim 1, wherein thedoped anion-cation complex is further configured to: provide nearinfrared fluorescence for optical imaging; or provide nuclear contrastfor medical imaging.
 3. The particle formulation of claim 2, wherein thecomplex is configured to: provide near infrared fluorescence emission ata 650-1000 nm spectral region, by doping (D) with an organic moleculeselected from the group consisting of indocyanine green and fluorescein,at levels from 0.0001 to 50 weight % of the complex; or provide nuclearcontrast for one or more of single photon emission computed tomography,positron emission tomography (SPECT/PET), or radionuclide mediatedtherapy by surface labelling with a radionuclide selected from the groupconsisting of ¹⁵³Sm, ⁹⁹mTc, ¹²³I, ¹¹¹In, ¹⁸⁸Re, ¹⁶⁶Ho, ⁹⁰Y, ⁸²Rb, ²²⁵Ac,²¹¹At, ²¹²Bi, ²²³Ra, ²¹²Pb, ²²⁷Th, and ¹⁴⁹Tb.
 4. The particleformulation of claim 1, wherein the calcium cation of the dopedanion-cation complex is derived from a compound selected from the groupconsisting of beta-tricalcium phosphate (Ca₃(PO₄)₂), calcium dihydrogenphosphate (Ca(H₂PO₄)₂), calcium hydrogen phosphate (CaHPO4), monocalciumphosphate monohydrate (Ca(H₂PO₄)·H₂O), dicalcium phosphate dihydrate(CaHPO₄·2H₂O), tetracalcium phosphate (Ca₄(PO₄)₂O), octacalciumphosphate (Ca₈H₂PO₄)₆·5H₂O), fluoroapatite (Ca₅(PO₄)₃F), chlorapatite(Ca₅(PO₄)₃C1), and a calcium phosphosilicate comprising 35-65 wt % ofSiO₂, 1-50 wt % of Na₂O, 10-90 wt % of CaO, and 1-50 wt % of P₂O₅, andcombinations thereof.
 5. The particle formulation of claim 1, whereinthe heat generated is up to 100° C. on exposure to a radiofrequencyfield of a frequency ranging from 1 Hz to 100 GHz and power in a range 1to 1000 W for a time period ranging from 0.1 second to 1 hour.
 6. Theparticle formulation of claim 5, wherein the heat generated is up to100° C. on exposure to the radiofrequency field of a frequency of 314kHz and the power of 100 W for the time period of 1 hour.
 7. Theparticle formulation of claim 1, wherein the formulation is selectedfrom one of (CaFe_(x)Mo_(y))₃(PO₄)₂, (CaFe_(x)Mo_(y))₁₀(PO₄)₆(OH)₂, or(CaFe_(x)Mo_(y))NaO₆PSiO₄, where x for Fe varies from 1.5 to 6 atomic %relative to Ca²⁺, and y for Mo varies from 0.0001 to 50 atomic %relative to Ca²⁺.
 8. The particle formulation of claim 1, wherein theparticles have a spherical shape.
 9. The particle formulation of claim8, wherein the particles have an average size in a range of 150 ±100 nm.10. The particle formulation of claim 1, further loaded with therapeuticagents for radio-wave-triggered controlled drug release.
 11. Aradio-wave responsive micro-bead formulation, comprising: a dopedanion-cation complex represented as D-C X, wherein: C is calcium cation(Ca²⁺); X is an anion selected from the group consisting of phosphate,pyrophosphate, and phosphosilicate; and D is a dopant comprising 0.0001to 50 atomic % of Mo and 1.5 to 6 atomic % of Fe, relative to Ca²⁺;wherein the complex is configured to: generate heat under exposure toradiofrequency (RF) waves; provide simultaneous T1 and T2 contrast undermagnetic resonance imaging (MRI); and provide X ray contrast under CTimaging, wherein the micro-bead formulation is selected from one or moreformulations selected from the group consisting of: a radio-waveresponsive, MR, CT, nuclear and/or NIR imageable micro-bead formulationranging in size from 1 μm to 1 mm for vascular embolization or tissueimplantation; a radio-wave responsive, MR, CT, nuclear and/or NIRimageable micro-bead formulation labelled with radioisotopes forradio-embolization therapy; and a radio-wave responsive, MR, CT, nuclearand/or NIR imageable formulation for culturing, proliferating,differentiating, activating, or reprogramming biological cells fortherapeutics.
 12. The micro-bead formulation of claim 11, wherein theformulation is a radio-wave responsive, MR-CT contrast enabled, andmicro-beads in the micro-bead formulation have a size in a range of 200±100 μm, for embolization or tissue implantation.